Measuring blood cell dynamics within the capillaries of the living eye provides crucial information regarding the health of the microvascular network. To date, the study of single blood cell movement in this network has been obscured by optical aberrations, hindered by weak optical contrast, and often required injection of exogenous fluorescent dyes to perform measurements. Here we present a new strategy to non-invasively image single blood cells in the living mouse eye without contrast agents. Eye aberrations were corrected with an adaptive optics camera coupled with a fast, 15 kHz scanned beam orthogonal to a capillary of interest. Blood cells were imaged as they flowed past a near infrared imaging beam to which the eye is relatively insensitive. Optical contrast of cells was optimized using differential scatter of blood cells in the split-detector imaging configuration. Combined, these strategies provide label-free, non-invasive imaging of blood cells in the retina as they travel in single file in capillaries, enabling determination of cell flux, morphology, class, velocity, and rheology at the single cell level.

Tissue light scatter limits the visualization of the microvascular network deep inside the living mammal. The transparency of the mammalian eye provides a noninvasive view of the microvessels of the retina, a part of the central nervous system. Despite its clarity, imperfections in the optics of the eye blur microscopic retinal capillaries, and single blood cells flowing within. This limits early evaluation of microvascular diseases that originate in capillaries. To break this barrier, we use 15 kHz adaptive optics imaging to noninvasively measure single-cell blood flow, in one of the most widely used research animals: the C57BL/6J mouse. Measured flow ranged four orders of magnitude (0.0002–1.55 µL min–1) across the full spectrum of retinal vessel diameters (3.2–45.8 µm), without requiring surgery or contrast dye. Here, we describe the ultrafast imaging, analysis pipeline and automated measurement of millions of blood cell speeds.

Abstract:The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina.

Offset aperture and split detector imaging are variants of adaptive optics scanning ophthalmoscopy recently introduced to improve the image contrast of retinal cells. Unlike conventional confocal scanning ophthalmoscopy, these approaches collect light laterally decentered from the optical axis. A complete explanation of how these methods enhance contrast has not been described. Here, we provide an optical model with supporting in vivo data that shows contrast is generated from spatial variations in refractive index as it is in phase contrast microscopy. A prediction of this model is supported by experimental data that shows contrast is optimized when the detector is placed conjugate with a deeper backscattering screen such as the retinal pigment epithelium and choroid, rather than with the layer being imaged as in conventional confocal imaging. This detection strategy provides a substantial improvement in the contrast these new methods can produce.

Purpose To characterize the early structural and functional changes in the retinal microvasculature in response to hyperglycemia in the Ins2 Akita mouse. Methods A custom phase-contrast adaptive optics scanning light ophthalmoscope was used to image retinal capillaries of 9 Ins2 Akita positive (hyperglycemic) and 9 Ins2 Akita negative (euglycemic) mice from postnatal weeks 5 to 18. A 15 kHz point scan was used to image capillaries and measure red blood cell flux at biweekly intervals; measurements were performed manually. Retinal thickness and fundus photos were captured monthly using a commercial scanning laser ophthalmoscope/optical coherence tomography. Retinal thickness was calculated using a custom algorithm. Blood glucose and weight were tracked throughout the duration of the study. Results Elevated blood glucose (>250 mg/dL) was observed at 4 to 5 weeks of age in Ins2 Akita mice and remained elevated throughout the study, whereas euglycemic littermates maintained normal glucose levels. There was no significant difference in red blood cell flux, capillary anatomy, lumen diameter, or occurrence of stalled capillaries between hyperglycemic and euglycemic mice between postnatal weeks 5 and 18. Hyperglycemic mice had a thinner retina than euglycemic littermates ( p < 0.001), but retinal thickness did not change with duration of hyperglycemia despite glucose levels that were more than twice times normal. Conclusions In early stages of hyperglycemia, retinal microvasculature structure (lumen diameter, capillary anatomy) and function (red blood cell flux, capillary perfusion) were not impaired despite 3 months of chronically elevated blood glucose. These findings suggest that hyperglycemia alone for 3 months does not alter capillary structure or function in profoundly hyperglycemic mice.